Nucleic acid fluorescent dye as well as preparation and application thereof
A fluorescent dye and compound technology, applied in the field of fluorescent dyes, can solve the problems of low sequence preference, impact on PCR, and difficulty in synthesis, and achieve the effects of low sequence preference, high sensitivity detection, and excellent fluorescence performance
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[0051] Fluorescent dye of the present invention can adopt following preparation method to make, and preparation method comprises the following steps:
[0052] 1) First, methylate 2-methylmercaptobenzothiazole (with or without substituents) with a methylating reagent (such as methyl iodide) to obtain compound a
[0053] 2) 2-hydroxy-4-methylquinoline and phenylboronic acid are catalyzed by copper acetate to obtain compound b
[0054] 3) Will Mixed with phosphorus oxychloride to give the compound It is then combined with the compound mix to get compound It reacts with N,N-dimethyl-1,3-diaminopropane to give the final dye structure
[0055] Concrete reaction formula is as follows:
[0056]
Embodiment 1
[0058] Embodiment 1: Preparation of nucleic acid fluorescent dye of the present invention
[0059] The preparation method is as follows:
[0060] 1) Dissolve the raw material 2-methylmercaptobenzothiazole (1.5g, 8.3mmol, 1eq) in 30mL of dry ethanol, add iodomethane (1.4g, 10mmol, 1.2eq), and react at 60°C for 5 hours. After the reaction was completed, the solvent was removed, washed several times with petroleum ether, and the off-white solid product a was collected by suction filtration:
[0061]
[0062] 2) The raw material 2-hydroxyl-4-methylquinoline (3.0g, 18.8mmol, 1eq) and phenylboronic acid (2.3g, 18.8mmol, 1eq) and copper acetate (3.75g, 18.8mmol, 1eq) were dissolved in 150 -250mL of dichloromethane, stirred and reacted at room temperature for 48-96h, then filtered under reduced pressure. Extract the filtrate with water three times, keep the dichloromethane phase extract, add anhydrous calcium chloride to dry for 1-2h. Concentrate the extract, pass through SiO 2...
Embodiment 2
[0066] Embodiment 2: Absorption spectrum and fluorescence spectrum of compound c
[0067] The DNA samples were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and were two complementary oligonucleotide chains, as shown in Table 1. The DNA samples were purified by HPLC, and an appropriate amount of DNA was dissolved in TE buffer (pH 7.4, 10mmol / LTris, 1mmol / L EDTA) according to the instructions to prepare a stock solution of corresponding concentration, and stored at 4°C. When in use, the above two single strands (Pro-20 and Tar-20) at the same concentration can be added to form double-stranded DNA (dsDNA-1).
[0068] Table 1
[0069]
[0070] The absorption spectrum of compound c (1 μmol / L) and the fluorescence emission spectrum combined with dsDNA-1 (20bp, 100nmol / L) were detected in TE buffer ( figure 1 shown). The maximum absorption peak of compound c is 467nm (blue light), and the fluorescence maximum emission peak after binding to DNA is located at 505nm ...
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